Anti-CD28 Humanized Antibodies

ABSTRACT

The invention relates to humanized antibodies directed against the human lymphocyte receptor CD28. When used in a monovalent form these antibodies are antagonists, i.e. capable of blocking of the CD28/B7 interaction, without activating CD28. These antibodies can be used in particular as therapeutic agents for blocking T cell activation through the CD28 receptor.

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The present invention relates to humanized antibodies binding CD28, tomonovalent fragments thereof, and to their therapeutic uses, inparticular in the context of regulating T cell activation.

Abnormal activation of T cells is involved in the pathogenesis of manyautoimmune diseases, and also in transplant rejection phenomena, wherethey cause an immune response directed against the transplanted organ todevelop.

One of the most important systems for regulating T lymphocyte activationis the molecular system B7/CD28/CTLA4. This system plays, for example,an essential role in the mechanisms of transplant rejection (WOODWARD etal., Transplantation, 66, 14-20, 1998). The molecules B7.1 (CD80) andB7.2 (CD86) borne by the APCs can activate the receptor CD28 and alsothe receptor CTLA4 of T lymphocytes. The activation of CD28 sends the Tlymphocyte a positive signal which stimulates the cell; on the otherhand, the activation of CTLA4 sends a negative signal which leads to anon-response (anergy) (FALLARINO et al., J. Exp. Med., 188, 205-210,1998).

Resting T lymphocytes express a large amount of CD28 and very littleCTLA4. When there is a first cognitive contact between an APC and a Tlymphocyte, the CD28/B7 interaction is favored, which activates thecell. It is only several hours after the initiation of activation that,due to the increase in membrane expression of CTLA4, the affinity ofwhich for B7 is 5 to 10 times greater than that of CD28, the B7/CD28interaction shifts in favor of a B7/CTLA4 interaction.

Regulatory T lymphocytes express a large amount of CD28 and of CTLA4that prevent or allow, respectively, the suppressive activity ofregulatory T lymphocytes. In the presence of APC expressing high levelof B7, the CD28/B7 interaction prevents the suppressive activity ofregulatory T lymphocytes (Sansom et al., Trends Immunol. 24, 314-319,2003).

Selective inhibition of the agonist signal given to the T cell by CD28,leaving the antagonist system consisting of the pair CTLA4/B7 intact,via specific blocking of the CD28/B7 interaction, would make it possibleto prevent T lymphocyte activation and to promote immune suppression byregulatory T lymphocytes. Such specific blocking of the CD28/B7interaction can be obtained using some antibodies directed against CD28.

These antibodies are to be used in a monovalent form (for instance asFab or scFv fragments), since when used in their divalent native form,their binding to CD28 brings about the dimerization and the activationof this receptor. Fab fragments each contain a light chain and the firsthalf of a heavy chain; scFv fragments consist of the variable portionsof the heavy and light chains of a parent antibody, connected to oneanother via a flexible linker (CLACKSON et al., Nature, 352, 624-628,1991), thus forming a single-chain protein.

One such antibody is antibody CD28.3, produced by the hybridoma cellline CNCM I-2582, and disclosed in PCT application WO 02/051871. Thisantibody, when used in a monovalent form such as scFv fragments, iscapable of blocking in vitro the CD28 receptor without activating it(PCT WO 02/051871; VANHOVE et al., Blood, 102, 564-70, 2003), and hasshown also its efficiency in vivo in models of organ transplantation inmice and in primates (POIRIER et al., World Transplant Congress, Sydney,Australia. Aug. 16-21, 2008; POIRIER et al, Sci Trans Med, 2:17,p17ra10, 2010).

A drawback of all monoclonal antibodies derived from murine sources, istheir immunogenicity when administered to human subjects. They provokeanti-mouse immune response, which results in a lesser efficiency of thetreatment, in particular when repeated administration is required.

This drawback can, in principle, be avoided by the use of humanizedantibodies. The aim of humanization is to obtain a recombinant antibodywhich has similar antigen-binding properties as the mouse monoclonalantibody from which the complementarity-determining regions (CDRs)sequences were derived, and which is far less immunogenic in humans.

The CDRs are the portions of the variable domains of an antibody whichdirectly contact the antigen and determine the antigen-bindingspecificity; the framework regions (FRs) which are located between theCDRs in the variable domains do not directly contact the antigen, butserves as a scaffold to maintain the global structure of the variabledomains.

Several approaches to antibody humanization have been reported. The morewidely used are based on “CDR grafting”, which involves thetransplantation of the CDRs of a murine antibody into appropriate humanFRs. However, in many antibodies, some FR residues are important forantigen binding, because they influence the conformation of CDRs andthus their antigen binding properties, in particular the bindingaffinity. A loss in binding affinity is particularly detrimental in thecase of an antibody intended to be used in a monovalent form whichgenerally exhibit less affinity for the antigen than the native divalentantibody. Thus, in most cases, it is further necessary, in order toobtain a sufficient binding affinity, to reintroduce one or severalframework residues from the mouse antibody in the human FRs, with therisk of simultaneously bringing back unwanted immunogenicity.

Another approach to antibody humanisation, called “de-immunization”,involves the identification within the FRs regions of the antibody, ofB-cell and T-cell epitopes recognized as “foreign” and thereforepotentially immunogenic in humans, and to remove them by appropriateamino-acids substitutions. This approach however also entails the riskthat FR residues important for antigen binding are deleted. Moreover,some immunogenic epitopes may lie in the CDRs and trying to remove theminvolves a very high risk of destroying not only the antigen-bindingaffinity but also the antigen-binding specificity of the antibody.

Therefore, a major issue in antibody humanisation is to determine whichamino acid residues are critical for retaining the antigen-bindingproperties. Various methods have been proposed for predicting the moreappropriate sites for substitution in the FRs regions. Although theyprovide general principles that may be of some help in the first stepsof humanization, the final result greatly varies from an antibody toanother. Thus, for a given antibody, it is very difficult to foretellwhich substitutions will provide the desired result. In the case whereinnot only substitutions in the FRs, but also in the CDRs would benecessary to decrease satisfactorily the immunogenicity in humans, thefinal result becomes totally unpredictable.

The inventors have succeeded in producing humanized CD28.3 (hereinafterreferred to as hCD28.3), with a low immunogenicity, and which, althoughit has several amino-acids substitutions including a non-conservativeK→Q substitution in the CDR2 of the heavy chain, retains the CD28binding properties of the parent mouse CD28.3. When used in a monovalentform, the hCD28.3 of the invention also retains the CD28 bindingproperties of the parent mouse CD28.3.

The present invention provides an anti-CD28 antibody, characterised inthat it is selected among:

a) an antibody having a CD28-binding site consisting of:

-   -   a first variable domain (also defined herein as the “heavy chain        variable domain”) defined by the following sequence:

(SEQ ID NO: 1) VQLQQSGAELKKPGASVKVSCKASGYTFTEYIIHWIKLRSGQGLEWIGWFYPGSNDIQYNAQFKGKATLTADKSSSTVYMELTGLTPEDSAVYFCARRDD FSGYDALPYWGQGTLVTVSA,

-   -   wherein said variable domain may optionally further comprise a Q        residue at its N-terminal end;    -   a second variable domain (also defined herein as the “light        chain variable domain”) defined by the following sequence:

(SEQ ID NO: 2) DIQMTQSPSSLSASVGDRVTITCKTNENIYSNLAWYQQKDGKSPQLLIYAATHLVEGVPSRFSGSGSGTQYSLTISSLQPEDFGNYYCQHFWGTPXTFGG GTKLEIKR,

-   -   wherein X=C, A, or N.

b) an antibody having a CD28-binding site consisting of:

-   -   a first variable domain having the CDRs of the variable domain        of SEQ ID NO: 1;    -   a second variable domain having the CDRs of the variable domain        of SEQ ID NO: 2.

The term “anti-CD28 antibody” herein refers to any antigen-bindingprotein having at least one antigen-binding site (consisting of thevariable domains of the light chain and of the heavy chain) able tospecifically bind human CD28. It encompasses antibodies in a divalentform (such as native immunoglobulin molecules or F(ab)′₂ fragments) withtwo CD28-binding sites, as well as antibodies in a monovalent form whichhave a single CD28-binding site, (for instance Fab, Fab′, FAT and scFvfragments). In most cases, antibodies in a monovalent form will bepreferred.

It includes in particular recombinant antibodies comprising aCD28-binding site associated with one or more heterologouspolypeptide(s).

By way of example, an antibody of the invention may be a recombinant Fabor Fab′ fragment containing the constant domain CH1 of a humanimmunoglobulin fused at the C-terminal end of the variable domain of SEQID NO: 1, and the constant domain CL of a human immunoglobulin fused atthe C-terminal end of the variable domain of SEQ ID NO: 2. An example ofsuch a recombinant Fab fragment is a Fab fragment with a heavy chainhaving the sequence of amino-acids 21-251 of SEQ ID NO: 4 and a lightchain having the sequence of amino-acids 21-234 of SEQ ID NO: 6.

Also, a hCD28.3 antibody of the invention may comprise, besides thevariable domains of SEQ ID NO: 1 and SEQ ID NO: 2, defined above, one ormore of the following components:

-   -   a human constant region (Fc). This constant region can be        selected among constant domains from any class of        immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any        isotype, including IgG1, IgG2, IgG3 and IgG4. Preferred constant        regions are selected among constant domains of IgG, in        particular IgG4.    -   a protein which makes it possible to prolong the plasma        half-life when it is administered in vivo under monovalent form        as disclosed for instance in PCT WO 02/051871; in a preferred        embodiment, said protein is the CH2-CH3 domains of an IgG        molecule, as disclosed in PCT/IB/2010/000196; according to said        embodiment, a hCD28.3 monovalent antibody of the invention is an        heterodimer of:    -   a first protein chain consisting essentially of, from its        N-terminus to its C-terminus:    -   a region A having the sequence SEQ ID NO: 1;    -   a region B consisting of a peptide linker and the CH2 and CH3        domains of an IgG immunoglobulin;    -   a second protein chain consisting essentially of, from its        N-terminus to its C-terminus:    -   a region A′ having the sequence SEQ ID NO: 2;    -   a region B identical to the region B of the first polypeptide.

Preferably, the peptide linker is the hinge region of human IgG1immunoglobulins having the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 7), andthe CH2 and CH3 domains are those of an immunoglobulin of the IgG4subclass. One can also use a shortened version of said hinge region,having the sequence DKTHTCPPCP (SEQ ID NO: 8).

According to a preferred embodiment, the polypeptide sequence of thefirst protein chain is the sequence of amino-acids 21-368 of SEQ ID NO:10, and the polypeptide sequence of the second protein chain is thesequence of amino-acids 21-355 of SEQ ID NO: 12. According to anotherpreferred embodiment, the polypeptide sequence of the first proteinchain is the sequence of amino-acids 21-373 of SEQ ID NO: 14, and thepolypeptide sequence of the second protein chain is the sequence ofamino-acids 21-360 of SEQ ID NO: 16.

Optionally, a hCD28.3 antibody of the invention may further comprise oneor more of the following components:

-   -   a protein having pharmacological activity (for example a toxin);    -   one or more tag polypeptide(s).

Alternatively, to prolong their plasma half life, in particular whenthey are under the form of Fab fragments, the antibodies of theinvention can be conjugated with water soluble polymers such aspolyethylene glycol (PEGylation). PEGylation is a classical way toenhance the pharmacokinetic properties of therapeutic polypeptides, andcan be achieved by techniques known in the art.

In this respect, the inventors found that the replacement of theoriginal cysteine residue at position 96 of the variable domain of thenative CD 28.3 by an alanine or an asparagine residue (resulting in anantibody having a light chain containing a variable domain of SEQ ID NO:2 wherein X=A or N) allowed a better efficacy in pegylation of theantibody using maleimide-activated polyethylene glycol (targetingreactive cystein residues), without modifying substantially its bindingactivity, although cysteine-96 is comprised in the CDR3 of the antibodylight chain. The benefit of the replacement of the original cysteineresidue at position 96 of the variable domain mainly consist in aspecific branching of the polyethylene glycol onto the C-terminalcysteine residue of the heavy chain. Without replacement of the originalcysteine residue at position 96 of the variable domain of the native CD28.3, maleimide-activated polyethylene glycol can bind to that cysteineresidue and impair the binding activity of the Fab molecule.

The inventors also found that addition of a di-alanine extension afterthe C-terminal cysteine of the heavy chain also resulted in a betterpegylation efficiency.

The invention also encompasses a polynucleotide selected among:

a) a polynucleotide encoding a polypeptide having the CDRs of SEQ ID NO:1, in particular a polynucleotide encoding a polypeptide of SEQ ID NO:1;

b) a polynucleotide encoding a polypeptide having the CDRs of SEQ ID NO:2, in particular a polynucleotide encoding a polypeptide of SEQ ID NO:2;

c) a polynucleotide encoding an hCD28.3 antibody of the invention, asdefined above.

Polynucleotides of the invention generally also comprise additionalsequences: for instance they may advantageously comprise a sequenceencoding a leader sequence or signal peptide allowing secretion of saidprotein chain.

The present invention also encompasses recombinant vectors, inparticular expression vectors, comprising a polynucleotide of theinvention, associated with transcription- and translation-controllingelements which are active in the host cell chosen. Vectors which can beused to construct expression vectors in accordance with the inventionare known in themselves, and will be chosen in particular as a functionof the host cell intended to be used.

The present invention also encompasses host-cells transformed with apolynucleotide of the invention. Preferably, said host cell istransformed with a polynucleotide comprising a sequence encoding theheavy chain of a hCD28.3 antibody of the invention and a polynucleotidecomprising a sequence encoding the light chain of a hCD28.3 antibody ofthe invention, and expresses said antibody. Said polynucleotides can beinserted in the same expression vector, or in two separate expressionvectors.

Host cells which can be used in the context of the present invention canbe prokaryotic or eukaryotic cells. Among the eukaryotic cells which canbe used, mention will in particular be made of plant cells, cells fromyeast, such as Saccharomyces, insect cells, such as Drosophila orSpodoptera cells, and mammalian cells such as HeLa, CHO, 3T3, C127, BHK,COS, etc., cells.

The construction of expression vectors of the invention and thetransformation of the host cells can be carried out by the conventionaltechniques of molecular biology.

Still another objet of the invention is a method for preparing a hCD28.3antibody of the invention. Said method comprises culturing a host-celltransformed with a polynucleotide comprising a sequence encoding theheavy chain of a hCD28.3 antibody of the invention and a polynucleotidecomprising a sequence encoding the light chain of a hCD28.3 antibody ofthe invention and recovering said antibody from said culture.

If the antibody is secreted by the host-cell, it can be recovereddirectly from the culture medium; if not, cell lysis will be carried outbeforehand. The antibody can then be purified from the culture medium orfrom the cell lysate, by conventional procedures, known in themselves tothose skilled in the art, for example by fractionated precipitation, inparticular precipitation with ammonium sulfate, electrophoresis, gelfiltration, affinity chromatography, etc.

The hCD28.3 antibodies of the invention can be used to obtain medicinalproducts. These medicinal products are also part of the object of theinvention.

The present invention also comprises a therapeutic compositioncomprising a hCD28.3 antibody of the invention, together with apharmaceutically acceptable excipient.

Preferably, said composition is a composition for parenteraladministration, formulated to allow the administration of a dose of from0.5 to 20 mg/Kg, advantageously of from 5 to 10 mg/Kg of an hCD28.3antibody of the invention. The injection route of the composition can bepreferably sub-cutaneous or intra-venous.

For instance, hCD28.3 antibodies of the invention can be used to obtainimmunosuppressant medicinal products which selectively blocks T cellactivation phenomena involving the CD28 receptor. Such immunosuppressantmedicinal products which act by selective blocking of CD28 haveapplications in all T lymphocyte-dependent pathological conditions,including in particular transplant rejection, graft-versus-host disease,T lymphocyte-mediated autoimmune diseases, such as type I diabetes,rheumatoid arthritis or multiple sclerosis, and type IVhypersensitivity, which is involved in allergic phenomena and also inthe pathogenesis of chronic inflammatory diseases, in particularfollowing infection with a pathogenic agent (in particular leprosy,tuberculosis, leishmaniasis, listeriosis, etc.).

The present invention will be understood more clearly from the furtherdescription which follows, which refers to nonlimiting examples of thepreparation and properties of a hCD28.3 antibody in accordance with theinvention.

The construction of expression vectors of the invention and thetransformation of host-cells can be made by the standard techniques ofmolecular biology.

A hCD28.3 antibody of the invention can be obtained by culturing a hostcell containing an expression vector comprising a nucleic acid sequenceencoding said antibody, under conditions suitable for the expressionthereof, and recovering said antibody from the host cell culture.

The present invention will be further illustrated by the followingadditional description, which refers to examples illustrating theproperties of hCD28.3 antibodies of the invention. It should beunderstood however that these examples are given only by way ofillustration of the invention and do not constitute in any way alimitation thereof.

LEGENDS OF THE DRAWINGS

FIG. 1: Nucleotidic (SEQ ID NO: 3) and amino acid (SEQ ID NO: 4)sequences of the Signal-VH-hCH1 construction. Bold: leader sequence(Nucleotide, SEQ ID NO: 17; amino acid, SEQ ID NO: 18); Underlined:positions of the CDRs (SEQ ID NO: 19, 20 and 21) of the parent CD28.3antibody. Italics: human CH1 region (Nucleotide, SEQ ID

NO: 22; amino acid, SEQ ID NO: 23); Highlighted and double underlined:substitutions made in the CD28.3 antibody VH region.

FIG. 2: Nucleotidic (SEQ ID NO: 5) and amino acid (SEQ ID NO: 6)sequences of the Signal-VL-hCκ construction. Bold: leader sequence(Nucleotide, SEQ ID NO: 24; amino acid, SEQ ID NO: 25); Underlined:positions of the CDRs (SEQ ID NO: 26, 27, and 28) of the parent CD28.3antibody. Italics: human c kappa region (Nucleotide, SEQ ID NO: 29;amino acid, SEQ ID NO: 30); Highlighted and double underlined:substitutions made in the CD28.3 antibody VL region.

FIG. 3: A) optical density at 405 nm for increasing concentrations ofFR104, hCD28.3 Fab or CD28.3 Fab in the Binding ELISA; B) calculation ofthe regression curves, allowing for determining comparative AC50 values.

FIG. 4: Nucleotidic (SEQ ID NO: 9) and amino acid (SEQ ID NO: 10)sequences of the hVHCD28.3-short hingeγ1-hγ4CH2CH3 construction. Bold:leader sequence (SEQ ID NO: 18). Underlined: CDRs (SEQ ID NO: 19, 20,and 21). Double underlined: hinge region (SEQ ID NO: 8). Dottedunderlined: CH2-CH3 domains (SEQ ID NO: 31) of the human IgG4.

FIG. 5: Nucleotidic (SEQ ID NO: 11) and amino acid (SEQ ID NO: 12)sequences of the hVLCD28.3-short hingeγ1-hγ4CH2CH3 construction. Bold:leader sequence (SEQ ID NO: 25). Underlined: CDRs (SEQ ID NO: 26, 27 and28). Double underlined: hinge region (SEQ ID NO: 8). Dotted underlined:CH2-CH3 domains (SEQ ID NO: 31) of the human IgG4.

FIG. 6: Nucleotidic (SEQ ID NO: 13) and amino acid (SEQ ID NO: 14)sequences of the hVHCD28.3-full hingeγ1-hγ4CH2CH3 construction. Bold:leader sequence (SEQ ID NO: 18). Underlined: CDRs (SEQ ID NO: 19, 20,and 21). Double underlined: hinge region (SEQ ID NO: 7). Dottedunderlined: CH2-CH3 domains (SEQ ID NO: 31) of the human IgG1.

FIG. 7: Nucleotidic (SEQ ID NO: 15) and amino acid (SEQ ID NO: 16)sequences of the hVLCD28.3-full hingeγ1-hγ4CH2CH3 construction. Bold:leader sequence (SEQ ID NO: 25). Underlined: CDRs (SEQ ID NO: 26, 27 and28). Double underlined: hinge region (SEQ ID NO: 7). Dotted underlined:CH2-CH3 domains (SEQ ID NO: 31) of the human IgG1.

FIG. 8: Anti-CD28 binding properties of hVH/VL CD28.3 monovalentantibodies. COS cells were co-transfected with 2 μg (each)pSignal-hVH-short hingeγ1-hγ4CH2-CH3 and pSignal-hVL-shorthingeγ1-hγ4CH2-CH3, or co-transfected with 2 μg (each) pSignal-hVH-fullhingeγ1-hγ4CH2-CH3 and pSignal-hVL-full hingeγ1-hγ4CH2-CH3. After 6days, supernatants were collected and monovalent antibodies were dosedusing a first sandwich ELISA. Supernatants were also assessed with abinding ELISA on immobilized CD28 target molecules and bound monovalentanti-CD28 antibodies were revealed with anti-human Fc antibodies labeledwith peroxidase. A: Optical density obtained with indicated moleculesaccording to their concentration. B: table with regression curves andthe calculation of ED50 (effective dose 50), the concentration needed toreach 50% binding activity in this assay.

FIG. 9: hVH/VL CD28.3 monovalent antibodies inhibit IL-2 secretion byactivated T cells. Jurkat T cells were stimulated with SEE superantigenand Raji antigen-presenting-cells during 48 h, in the presence ofindicated concentrations of purified hVH/VL-short hingeγ1-hγ4CH2-CH3monovalent antibodies. Supernatant were collected and IL-2 measured byELISA.

FIG. 10: SP sepharose HP-chromatography (left) and SDS-PAGE (right)under unreduced conditions after pegylation of C96-Fabs from humanisedCD28.3 antibody. Lane 1: marker; lane 2:load; lane 3: Peak 1; lane 4:Peak 2; lane 5: Peak 3.

FIG. 11: Binding properties for CD28 of recombinant hCD28.3 Fabs with orwithout C96 mutations. The graph shows binding activity (Y axis)according to Fab concentration (X axis).

FIG. 12: SP sepharose HP-chromatograpghy (left) and SDS-PAGE (right)under unreduced conditions after pegylation of C96A-Fabs from humanisedCD28.3 antibody. Lane 1: MW markers; lane 2: Pegylated proteinspre-chromatography; lane 3: peak 1 containing the monopegylated Fab,representing 41% of the starting material.

FIG. 13: SP sepharose HP-chromatograpghy (left) and SDS-PAGE (right)under unreduced conditions after pegylation of C96A-Fabs from humanisedCD28.3 antibody with a CAA C-terminal sequence in the heavy chain. Lane1: MW markers; lane 2: Pegylated proteins pre-chromatography; lane 3:peak.

EXAMPLE 1 Construction and Eucaryotic Expression of A hCD28.3 MonovalentAntibody (Fab Fragment)

Heavy Chain:

The sequence encoding the VH region of hCD28.3 (SEQ ID NO: 1) in fusionwith the sequence encoding the human CH1 region (NCBI Accession numberAAF03881) and with a sequence encoding the leader peptide of the heavychain of the native murine CD28.3 antibody, was synthetized chemically,and introduced in the cloning vector pGA18 (Geneart) for amplification.The sequence was then excised by digestion with KpnI/BamHI restrictionenzymes and subcloned into the KpnI/BamHI sites of the plasmidpcDNA3.1-hygro (Invitrogen). Positive clones were amplified and purifiedby Midiprep-endotoxin free (Macherey-Nagel) for transfection step.

The resulting plasmid is designated pSignal-VH-hCH1. It comprises aconstruct containing the sequence encoding the VH region of hCD28.3between the sequence encoding the CD28.3 heavy chain leader peptide andthe sequence encoding the human CH1 region (NCBI Accession numberAAF03881).The nucleotidic and amino acid sequences of this construct areshown on FIG. 1. They are also represented as SEQ ID NO: 3 and SEQ IDNO: 4 in the enclosed sequence listing.

Light Chain:

The sequence encoding the VL region of hCD28.3 (SEQ ID NO: 2) in fusionwith the sequence encoding the human c kappa region (NCBI accessionnumber BAC01725) and with a sequence encoding the leader peptide of thelight chain of the native murine CD28.3 antibody, was synthetizedchemically, and introduced in the cloning vector pGA18 (Geneart) foramplification. The sequence was then excised by digestion withKpnI/BamHI restriction enzymes and subcloned into the KpnI/BamHI sitesof the plasmid pcDNA3.A-hygro (Invitrogen). Positive clones wereamplified and purified by Midiprep-endotoxin free (Macherey-Nagel) fortransfection step.

The resulting plasmid is designated pSignal-VL-hCκ. It comprises aconstruct containing the sequence encoding the VL region of hCD28.3between the sequence encoding the CD28.3 light chain signal peptide andthe sequence encoding the human c kappa region (NCBI accession numberBAC01725). The nucleotidic and amino acid sequences of this constructare shown on FIG. 2. They are also represented as SEQ ID NO: 5 and SEQID NO: 6 in the enclosed sequence listing.

Eucarvotic Expression

COS cells were co-transfected with 2 μg (each) pSignal-VL-hCH1 andpSignal-VH-hCH1 using the Fugene lipofection kit (Roche Diagnostics,Basel, Switzerland) according to the manufacturer's instructions.Cultures were maintained for 3 days at 37° C., divided one third, andput back into culture for an additional 3 days, after which time thecell supernatants were collected.

The activity of the hCD28.3 monovalent antibody is evaluated directly inthe supernatant by ELISA, as described in Example 2 below.

EXAMPLE 2 Detection of the hCD28.3 Fab Fragment Binding Activity byElisa

The binding properties of the hCD28.3 Fab fragment have been comparedwith those obtained after transfection of Cos cells with plasmids codingfor CD28.3 Fab (not humanized), using two ELISA assays

-   -   First (Sandwich ELISA), the concentrations of the hCD28.3 and        CD28.3 Fab fragments in the culture supernatants of transfected        COS cells have been determined using a sandwich ELISA. Briefly,        the anti-CD28 Fab contained in the supernatants are first        captured by a rabbit polyclonal antibody, specific for the heavy        and light variable domains of CD28.3 (obtained after        immunization of rabbits with a single-chain-Fv containing the        heavy and light variable domains of the native CD28.3, and        purified by immunoadsorption on CD28.3 Fab-Sepharose) . The        captured proteins are then revealed with a murine monoclonal        antibody directed to the kappa chain of human IgG, followed by a        polyclonal goat anti-mouse antibody labelled with peroxidase.        Bound antibody was revealed by colorimetry using the TMB        substrate, and read at 405 nm.

The OD corresponding to different dilutions of the supernatant are thencompared to a standard curve obtained with known quantities of a CD28.3Fab, called FR104, purified from culture supernatant of transformed CHOcells with standard techniques of chromatography, and dosed with a BCA(bisynchronic acid) assay. FR104 contains the native (not humanized), VHand VL regions of the CD28.3 antibody. Therefore, we can evaluate theamount of Fab proteins present in cell supernatants.

-   -   Second (Binding ELISA), for testing the binding activity of        hCD28.3 Fab fragments compared to CD28.3 Fab, chimeric human        CD28/Fc (R&D Systems, Abingdon, United Kingdom) was used at 2        μg/ml in carbonate buffer 0.05M pH 9.2 to coat the wells (50        μL/well) of microtiter plates (Nunc Immunoplates) overnight at        4° C. These immobilized CD28 target molecules will bind only        immunoreactive molecules with anti-CD28 activity.

The wells were then washed 3 times successively with 200 μL PBS-0.05%Tween, and saturated with 100 μL PBS Tween 0.1% BSA 1% for 2 hours at37° C.

Then, after 3 washings with 200 μL PBS-0.05% Tween, supernatantscontaining known concentrations of CD28.3 or hCD28.3 Fab fragments wereadded (50 μL/well) at different dilutions in PBS-0.1% Tween andincubated for 2 hours at 37° C. After 3 washings with 200 μL PBS-0.05%Tween, a murine monoclonal antibody directed to the kappa chain of humanIgG, (1/10000 dilution) was added (1 hour, 37° C.), followed byperoxidase-conjugated goat anti-mouse antibodies (1/2000 dilution),followed by colorimetric revelation using the TMB substrate and readingat 405 nm.

Then the results are plotted as the absorbance (Y axis), measured withthe binding ELISA, according to the Fab concentration (X axis), measuredwith the sandwich ELISA. An AC50 (Antibody Concentration 50) isdetermined after calculating the slope of the curve in its linear rangeas the concentration of the anti-CD28 Fab needed to reach 50% of themaximal optical density (OD) in the binding assay.

The results are shown on FIG. 3 and Table I.

Item A in FIG. 3 shows the optical density at 405 nm for increasingconcentrations of FR104, hCD28.3 Fab or CD28.3 Fab in the Binding ELISA.

Item B in FIG. 3 shows the calculation of the regression curves,allowing for determining comparative AC50 values.

Table I below summarises the OD50, the equation, and the AC50for thestandard FR104, and the Fab fragments VH-wild type +VL-wild type and FabhCD28.3

TABLE I OD50 Equation AC50 Std FR104 1.792 y = 1.1424Ln(x) − 3.6351 115CD28.3 Fab 1.82 y = 0.9776Ln(x) − 3.2483 162 hCD28.3 Fab 1.804 y =1.0217Ln(x) − 3.2859 151

These results show that 50% of the binding activity to CD28 could bereached at a concentration similar for Fab fragments VH-wild type+VL-wild type (CD28.3 Fab) and hCD28.3 Fab. The concentration isslightly lower for the standard, probably because it is purified beforethe assay. Thus hCD28.3 retains the CD28-binding properties of the wildtype VH and VL sequences of CD28.

EXAMPLE 3 Construction and Eucaryotic Expression of a hCD28.3 MonovalentAntibody (FV-FC Fragment) with a Short γ1 Hinge and a γ4 CH2-CH3 Domain

Heavy Chain:

The sequence encoding the VH region of hCD28.3 (SEQ ID NO: 1) inC-terminal fusion with the sequence encoding a portion of the hingeregion of the human IgG1 (SEQ ID NO: 8), with CH2-CH3 domains of thehuman IgG4 (nucleotides 787 to 1440 of the sequence NCBI Accessionnumber BC025985) and in N-terminal position with a sequence encoding theleader peptide of the heavy chain of the native murine CD28.3 antibody,was synthetized chemically, and introduced in the cloning vector pMA(Geneart) for amplification. The sequence was then excised by digestionwith NheI/EcoRI restriction enzymes and subcloned into the NheI/EcoRIsites of the plasmid pCIneo (Promega). After transformation of E. colicells, positive clones were amplified and extracted plasmids werepurified by Midiprep-endotoxin free columns (Macherey-Nagel).

The resulting plasmid is designated pSignal-hVH-shorthingeγ1-hγ4CH2-CH3.It comprises a construct containing the sequence encoding the VH regionof hCD28.3 between the sequence encoding the CD28.3 heavy chain signalpeptide and the sequence encoding a part of the human γ1 hinge regionand of the human γ4 CH2-CH3 domains. The nucleotidic and amino acidsequences of this construct are shown on FIG. 4. They are alsorepresented as SEQ ID NO: 9 and SEQ ID NO: 10 in the enclosed sequencelisting.

Light Chain:

The sequence encoding the VL region of hCD28.3 (SEQ ID NO: 2) in fusionwith the sequence encoding a portion of the hinge region of the humanIgG1 (SEQ ID NO: 8), with CH2-CH3 domains of the human IgG4 (nucleotides787 to 1440 of the sequence NCBI Accession number BC025985) and inN-terminal position with a sequence encoding the leader peptide of theheavy chain of the native murine CD28.3 antibody, was synthetizedchemically, and introduced in the cloning vector pMA (Geneart) foramplification. The sequence was then excised by digestion withNheI/EcoRI restriction enzymes and subcloned into the NheI/EcoRI sitesof the plasmid pCINeo (Promega). After transformation of E. coli cells,positive clones were amplified and extracted plasmids were purified byMidiprep-endotoxin free columns (Macherey-Nagel).

The resulting plasmid is designated pSignal-hVL-shorthingeγ1-hγ4CH2-CH3.It comprises a construct containing the sequence encoding the VL regionof hCD28.3 between the sequence encoding the CD28.3 light chain signalpeptide and the sequence encoding a part of the human γ1 hinge regionand of the human γ4 CH2-CH3 domains. The nucleotidic and amino acidsequences of this construct are shown on FIG. 5. They are alsorepresented as SEQ ID NO: 11 and SEQ ID NO: 12 in the enclosed sequencelisting. Eukaryotic expression

COS cells were co-transfected with 1 μg (each)pSignal-hVL-shorthingeγ1-hγ4CH2-CH3 andpSignal-hVH-shorthingeγ1-hγ4CH2-CH3, using the Lipofectamine lipofectionkit (Invitrogen) according to the manufacturer's instructions. Cultureswere maintained for 3 days at 37° C., after which time the cellsupernatants were collected. The activity of the monovalent antibody isevaluated directly in the supernatant by ELISA, as described in Example5 below.

EXAMPLE 4 Construction and Eucaryotic Expression of a hCD28.3 MonovalentAntibody (FV-FC Fragment) with a Full Length γ1 Hinge and a γ4 CH2-CH3Domain

Heavy Chain:

The sequence encoding the VH region of hCD28.3 (SEQ ID NO: 1) inC-terminal fusion with the sequence encoding a full length hinge regionof the human IgG1 (SEQ ID NO: 7) , with CH2-CH3 domains of the humanIgG4 (nucleotides 787 to 1440 of the sequence NCBI Accession numberBC025985) and in N-terminal position with a sequence encoding the leaderpeptide of the heavy chain of the native murine CD28.3 antibody, wassynthetized chemically, and introduced in the cloning vector pMA(Geneart) for amplification. The sequence was then excised by digestionwith NheI/EcoRI restriction enzymes and subcloned into the NheI/EcoRIsites of the plasmid pClneo (Promega). After transformation of E. colicells, positive clones were amplified and extracted plasmids werepurified by Midiprep-endotoxin free columns (Macherey-Nagel).

The resulting plasmid is designated pSignal-hVH-fullhingeγ1-hγ4CH2-CH3.It comprises a construct containing the sequence encoding the VH regionof hCD28.3 between the sequence encoding the CD28.3 heavy chain signalpeptide and the sequence encoding the human γ1 hinge region and thehuman γ4 CH2-CH3 domains. The nucleotidic and amino acid sequences ofthis construct are shown on FIG. 6. They are also represented as SEQ IDNO: 13 and SEQ ID NO: 14 in the enclosed sequence listing.

Light Chain:

The sequence encoding the VL region of hCD28.3 (SEQ ID NO: 2) in fusionwith the sequence encoding the full length hinge region of the humanIgG1 (SEQ ID NO: 7), with CH2-CH3 domains of the human IgG4 (nucleotides787 to 1440 of the sequence NCBI Accession number BC025985) and inN-terminal position with a sequence encoding the leader peptide of theheavy chain of the native murine CD28.3 antibody, was synthetizedchemically, and introduced in the cloning vector pMA (Geneart) foramplification. The sequence was then excised by digestion withNheI/EcoRI restriction enzymes and subcloned into the NheI/EcoRI sitesof the plasmid pCIneo (Promega). After transformation of E. coli cells,positive clones were amplified and extracted plasmids were purified byMidiprep-endotoxin free columns (Macherey-Nagel).

The resulting plasmid is designated pSignal-hVL-fullhingeγ1-hγ4CH2-CH3.It comprises a construct containing the sequence encoding the VL regionof hCD28.3 between the sequence encoding the CD28.3 light chain signalpeptide and the sequence encoding the human γ1 full length hinge regionand of the human γ4 CH2-CH3 domains. The nucleotidic and amino acidsequences of this construct are shown on FIG. 7. They are alsorepresented as SEQ ID NO: 15 and SEQ ID NO: 16 in the enclosed sequencelisting.

Eukaryotic Expression

COS cells were co-transfected with 1 μg (each)pSignal-hVH-fullhingeγ1-hγ4CH2-CH3 andpSignal-hVL-fullhingeγ1-hγ4CH2-CH3 plasmids using the Lipofectaminelipofection kit (Invitrogen) according to the manufacturer'sinstructions. Cultures were maintained for 3 days at 37° C., after whichtime the cell supernatants were collected.

The activity of the hCD28.3 monovalent antibody is evaluated directly inthe supernatant by ELISA, as described in Example 5 below.

EXAMPLE 5 Evaluation of the HCD28.3-Full Length γ1 Hinge-γ4CH2-CH3Domains and HCD28.3-Short γ1 Hinge-γ4CH2-CH3 Domains MonovalentAntibodies Binding Activity by Elisa

The binding properties of the hCD28.3 monovalent antibodies hCD28.3-fullγ1 hinge-γ4CH2-CH3 domains and hCD28.3-short γ1 hinge-γ4CH2-CH3 domainsproduced by transfected COS cells have been analysed using two ELISAassays.

-   -   First (Sandwich ELISA), the concentrations of the hCD28.3        monovalent antibodies in the culture supernatants of transfected        COS cells have been determined using a sandwich ELISA. Briefly,        the monovalent antibodies contained in the supernatants are        first captured by a goat polyclonal antibody directed to human        IgG. The captured proteins are then revealed with a biotinylated        goat polyclonal anti-human IgG, Fc specific, antibody, then, a        Peroxidase-conjugated streptavidin. Bound antibody was revealed        by colorimetry using the TMB substrate, and read at 405 nm.

The OD corresponding to different dilutions of the supernatant are thencompared to a standard curve obtained with known quantities of hCD28.3monovalent antibodies, purified from culture supernatant of transformedCHO cells with standard techniques of chromatography, and dosed with aBCA (bisynchronic acid) assay.

-   -   Second (Binding ELISA), for testing the binding activity of        hCD28.3 monovalent antibodies, chimeric human CD28/Fc (R&D        Systems, Abingdon, United Kingdom) was used at 2 μg/ml in        carbonate buffer 0.05M pH 9.2 to coat the wells (50 μL/well) of        microtiter plates (Nunc Immunoplates) overnight at 4° C. These        immobilized CD28 target molecules will bind only immunoreactive        molecules with anti-CD28 activity.

The wells were then washed 3 times successively with 200 μL PBS-0.05%Tween, and saturated with 100 μL PBS Tween 0.1% BSA 1% for 2 hours at37° C.

Then, after 3 washings with 200 μL PBS-0.05%Tween, supernatantscontaining known concentrations of the monovalent antibodies to betested were added (50 μL/well) at different dilutions in PBS-0.1% Tweenand incubated for 2 hours at 37° C. After 3 washings with 200 μLPBS-0.05% Tween, we added (1/500 dilution; 1 hour, 37° C.) a rabbitpolyclonal antiserum, specific for the heavy and light variable domainsof CD28.3 (obtained after immunization of rabbits with a single-chain-Fvcontaining the heavy and light variable domains of the native CD28.3,and purified by immunoadsorption on CD28.3 Fab-Sepharose). This wasfollowed by peroxidase-conjugated donkey anti-rabbit antibodies (1/2000dilution), followed by colorimetric revelation using the TMB substrateand reading at 405 nm.

Then the results are plotted as the absorbance (Y axis), measured withthe binding ELISA, according to the monovalent antibody concentration (Xaxis), measured with the sandwich ELISA. An AC50 (Antibody Concentration50) is determined after calculating the slope of the curve in its linearrange as the concentration of the monoclonal antibody needed to reach50% of the maximal optical density (OD) in the binding assay.

FIG. 8 compares binding activities of hCD28.3-full IgG1hinge-IgG4CH2-CH3 domains with hCD28.3-short IgG1 hinge-IgG4CH2-CH3domains monovalent antibodies in the Binding ELISA (item A in FIG. 8).

Item B in FIG. 8 summarises the equation, the regression factor and theAC50 for the monovalent antibodies.

These results show that 50% of the binding activity to CD28 could bereached at a concentration similar for hCD28.3-full γ1 hinge-γ4CH2-CH3domains or hCD28.3-short γ1 hinge-γ4CH2-CH3 domains monovalentantibodies.

EXAMPLE 6 hCD28.3 Monovalent Antibodies Prevents T Cell Activation

To verify that hCD28.3 monovalent antibody blocks CD28-dependent T cellactivation, we stimulated human T cells (Jurkat cells) with SEEsuperantigen presented by a Raji B cell line. The endotoxin SEE, whenpresented to the class II-positive B cell lymphoblastoid line Raji,activates the Vβ8-expressing T cell line Jurkat to secrete IL-2 (Hermanet al, 1990, J. Exp. Med. 172:709). Since Jurkat cells express highlevel of CD28 and Raji cells express CD80/86, this reaction is partiallydependant on CD28. We measured synthesis of interleukin-2 in this assayby ELISA (ELISA Max™ Set Deluxe Human IL-2 Kit; Biolegend #431805) after48 h, in the presence of increasing concentrations of hVH/VLCD28.3-short γ1 hinge-γ4CH2-CH3 domains.

The results are shown on FIG. 9. They reveal that hCD28.3 monovalentantibodies reduce IL-2 synthesis by T cells in a dose-dependent manner.

EXAMPLE 7 Preparation of a Pegylated hCD28.3 Monovalent Antibody

A hCD28.3 Fab fragment prepared as described in Example 1 was pegylatedwith maleimide-activated 40 KDa PEG using standard conditions forreduction and PEGylation.

Briefly, Fab antibody fragments were concentrated to 1 mg/mL and thendiafiltrated against 20 mM Sodium phosphate, 2 mM EDTA, and pH 7.0. Fab′antibody fragments were then reduced by addition of cysteamine chloridein a molar equivalent ratio=30:1 at room temperature. After 5 hours,solution was applied on a desalting column. Polyethylene glycol (PEG)(Sunbright GL2 400MA, NOF Corporation) was dissolved in 20 mM Phosphate,2 mM EDTA, pH 7.0 to give 9% (w/w) solution. Desalted Fab solution andPEG were mixed in a molar equivalent ratio=1:1.5 and incubated atambient temperature for 3 h. Following PEGylation the Fab-peg waspurified by chromatography using SP Sepharose HP medium. The targetprotein was eluted with a salt gradient from 0 to 1 M NaCl. Eluted peakswere analysed by SDS-Page. Peak 1 represented monopegylated material,peak 2 unpegylated material and peak 3 polypegylated material.

The results are shown on FIG. 10.

These results show that a significant part of the Fab proteins from theCD28.3 mAb presents a perturbed pegylation profile which results in ayield of monopegylated Fabs of about 5% only (peak 1).

The CD28.3 mAb contains a cystein residue (C96) that is not engaged inintra or inter-chain disulfide bridges, at position 96 of the variabledomain light chain. Free cystein will possess a higher reactivity thancystein residues engaged into disulfide bridges and will thereforepreferentially be targets of maleimide-activated pegs. Therefore it islikely that a second, unwanted pegylation occurs on this residue.

To solve that problem we performed a VL-C96 mutation study to determinewhether it was possible to substitute the C96 residue by another aminoacid without modifying the binding properties of the antibody.

Plasmid coding for humanized anti-CD28.3 Fabs with unmodified C96 in thelight chain, or with C96 to A, G, S, V, T, N or R mutations wereconstructed and transfected into Cos cells by lipofection, as disclosedin Example 1. Cell supernatants were first analyzed by sandwich ELISA todetermine total Fab concentration, as disclosed in Example 2. Thensupernatants were analysed by ELISA to determine binding activity onimmobilized recombinant CD28, as disclosed in Example 2.

The results are shown in FIG. 11. These results show that unlike allother substitutions tested, the C96A substitution resulted in a fullyactive antibody and that the C96N substitution resulted only in amoderate reduction of activity.

The C96A Fab fragment variant was pegylated and purified bychromatography as described above. Pegylated proteins pre-chromatographyand elution peaks were analysed by SDS-Page. The results are shown onFIG. 12. Peak 1 represents monopegylated material.

These results show that the C96A Fab fragment can be pegylated with anefficacy reaching 41% (FIG. 12).

Advantage of the CAA C-terminal end of the heavy chain. The immediatemolecular environment of a free cystein might modify its accessibilityto maleimide-pegylation and therefore modify the yield of the pegylationreaction. One possible option for the C-terminal cystein is to be thelast amino acid of the heavy chain. Another option is the addition of“stuff amino acids” at the C-terminal position, after the last cysteine.We therefore compared pegylation efficacy of a Fab′ molecule from theC96A variant of the humanized CD28.3 Mab with the last C-terminalcystein being the last amino acid of the heavy chain (C variant; datashown in FIG. 12) with a similar molecule with the last C-terminalcystein being followed by two alanins (CAA variant). Our data clearlyand reproducibly demonstrated that the CAA variant could be pegylatedwith a 20% higher efficacy (FIG. 13). Indeed pegylation yield that wasof 41% for the C96A-C variant reached 52% for the C96A-CAA variant.

1-11. (canceled)
 12. A method of treating a T-lymphocyte-mediatedchronic inflammatory disease in a subject in need thereof comprisingadministering an anti-CD28 monovalent antibody to the subject, whereinthe anti-CD28 monovalent antibody is selected from the group consistingof: (a) an antibody having a CD28-binding site consisting of: a heavychain variable domain of SEQ ID NO: 1; and a light chain variable domainof SEQ ID NO: 2, and (b) an antibody having a CD28-binding siteconsisting of: a heavy chain variable domain having all threecomplementarity determining regions (CDRs) of the variable domain of SEQID NO: 1; and a light chain variable domain having all three CDRs of thevariable domain of SEQ ID NO:
 2. 13. The method of claim 12, wherein themonovalent antibody is a heterodimer of: a first protein chain havingthe sequence of amino-acids 21-251 of SEQ ID NO: 4; and a second proteinchain having the sequence of amino-acids 21-234 of SEQ ID NO:
 6. 14. Themethod of claim 12, wherein the monovalent antibody is a heterodimer of:(I) a first protein chain consisting essentially of, from its N-terminusto its C-terminus: i: a region A which is a heavy chain variable domainof SEQ ID NO: 1; and ii: a region B consisting of a peptide linkerfollowed by the CH2 and CH3 domains of an IgG immunoglobulin, and (II) asecond protein chain consisting essentially of, from its N-terminus toits C-terminus: i: a region A′ which is a light chain variable domain ofSEQ ID NO: 2; and ii: a region B identical to the region B of the firstprotein chain.
 15. The method of claim 14, wherein the peptide linker isselected from the group consisting of: a peptide of SEQ ID NO: 7; and apeptide of SEQ ID NO:
 8. 16. The method of claim 14, wherein the CH2 andCH3 domains are those of an immunoglobulin of the IgG4 subclass.
 17. Themethod of claim 16, wherein the monovalent antibody is selected from thegroup consisting of: a monovalent antibody wherein the polypeptidesequence of the first protein chain is the sequence of amino-acids21-368 of SEQ ID NO: 10, and the polypeptide sequence of the secondprotein chain is the sequence of amino-acids 21-355 of SEQ ID NO: 12;and a monovalent antibody wherein the polypeptide sequence of the firstprotein chain is the sequence of amino-acids 21-373 of SEQ ID NO: 14,and the polypeptide sequence of the second protein chain is the sequenceof amino-acids 21-360 of SEQ ID NO:
 16. 18. The method of claim 13,wherein the second protein chain comprises a variable domain of SEQ IDNO:
 2. 19. The method of claim 12, wherein the monovalent antibody isadministered in a composition and the composition further comprises apharmaceutically acceptable excipient.
 20. The method of claim 13,wherein the second protein chain comprises a variable domain of SEQ IDNO: 2 where X represents an alanine or an asparagine residue.
 21. Themethod of claim 13, wherein the monovalent antibody is pegylated. 22.The method of claim 12, wherein the heavy chain variable domain furthercomprises a Q residue at the N-terminal end.
 23. The method of claim 12,wherein the T-lymphocyte-mediated chronic inflammatory disease istransplant rejection.
 24. The method of claim 12, wherein theT-lymphocyte-mediated chronic inflammatory disease is graft versus hostdisease.
 25. The method of claim 12, wherein the T-lymphocyte-mediatedchronic inflammatory disease is chronic allograft vasculopathy.
 26. Themethod of claim 12, wherein the T-lymphocyte-mediated chronicinflammatory disease is hypertension.